Compositions and methods for virus inhibition

ABSTRACT

The present invention provides compositions and methods for treating a measles virus infection. A pharmaceutical composition comprises a polypeptide in a biocompatible pharmaceutical carrier, in which the polypeptide consists of at least a portion of SEQ ID NO: 5 or SEQ ID NO: 6. A method embodiment comprises administering the polypeptide (preferably in a biocompatible pharmaceutical carrier) to a subject suffering from a measles infection.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 13/962,524,filed on Aug. 8, 2013, which is a division of U.S. application Ser. No.13/482,388, filed on May 29, 2012, now U.S. Pat. No. 8,598,116, which isa division of U.S. application Ser. No. 12/378,558, filed on Feb. 17,2009, now abandoned, which is a continuation of U.S. Ser. No.10/578,013, filed on May 3, 2006, now U.S. Pat. No. 7,491,793, which isthe National Stage of PCT/US2004/36578, filed on Nov. 3, 2004, whichclaims the benefit of U.S. Provisional Application Ser. No. 60/517,181,filed Nov. 4, 2003, each of which is incorporated herein by reference inits entirety.

FIELD OF THE INVENTION

The present invention relates to a method of preventing or inhibitingviral infection of a cell and/or fusion between the envelope of a virusand the membranes of a cell targeted by the virus (thereby preventingdelivery of the viral genome into the cell cytoplasm, a step requiredfor viral infection). The present invention provides methods foridentifying a fusion initiation region, or FIR, of the viruses. Thepresent invention provides for a method of identifying the FIR in theseviruses. The present invention further provides for methods ofpreventing infection by a Type I virus by interfering with its FIR.

INCORPORATION OF SEQUENCE LISTING TEXT FILE

This application includes biological sequence information in a SequenceListing presented in an ASCII text file named “TU-271-DIV-1-SEQ.txt”,created on Apr. 12, 2012, and having a file size of 23,569 bytes, whichis incorporated herein by reference.

INTRODUCTION

All viruses must bind to and invade their target cells to replicate. Forenveloped animal viruses, including RNA viruses having Class I membranefusion proteins (Type I viruses), the process involves (a) binding ofthe virion to the target cell, (b) fusion of the envelope of the viruswith the plasma membrane or an internal cellular membrane, (c)destabilisation of the viral envelope and cellular membrane at the fusedarea to create a fusion pore, (d) transfer of the viral RNA through thepore, and (e) modification of cellular function by the viral RNA.

Fusion of the viral membrane and the cell envelope, steps (b) and (c)above, is mediated by the interaction of a viral transmembraneglycoprotein (fusion protein) with surface proteins and membranes of thetarget cell. These interactions cause conformational changes in thefusion protein that result in the insertion of a viral fusion peptideinto the target cell membrane. This insertion is followed by furtherconformational changes within the fusion protein that bring the viralenvelope and cell membranes into close proximity and results in thefusion of the two membrane bilayers.

A virus is unable to spread and propagate within its host if this fusionprocess is disrupted. Intentional disruption of this fusion process canbe achieved by directing peptides and peptide mimics homologous tofusion protein sequences, antibodies that recognize the fusion protein,and other factors that act against the fusion protein.

BACKGROUND OF THE INVENTION

Structural Similarities Among RNA Virus Class I Fusion Proteins.

Hemagglutinin 2 (HA2) of influenza virus, an orthomyxovirus, is theprototypic RNA virus Class I fusion protein and contains an aminoterminal hydrophobic domain, referred to as the fusion peptide, that isexposed during cleavage of the hemagglutinin precursor protein. Themembrane fusion proteins of RNA viruses from several diverse families,including arenaviruses, coronaviruses, filoviruses, orthomyxoviruses,paramyxoviruses, and retroviruses, share several common structuralfeatures with HA2 and have been referred to as Class I viral fusionproteins.

It has been observed that the fusion protein of HIV-1, the transmembraneglycoprotein and other retroviral transmembrane proteins, like those oforthomyxoviruses and paramyxoviruses, possess a hydrophobic fusionpeptide domain exposed during cleavage of a precursor (gp160) (Gallaher,1987; Gonzalez-Scarano et al., 1987). Based on these similarities andcomputer algorithms that predict protein configurations, it has beensuggested (Gallaher et al., 1989) that the external portion (ectodomain,amino terminus) of HIV-1 transmembrane protein and the transmembraneproteins of other retroviruses, all could fit the scaffold of HA2structure as determined by x-ray crystallography (Wilson, Skehel, andWiley, 1981).

Based on these observations, it was predicted that retroviraltransmembrane proteins contain several structural features in additionto the fusion peptide in common with the known structure of HA2,including an extended amino terminal helix (N-helix, usually a “heptadrepeat” or “leucine zipper”), a carboxyl terminal helix (C-helix), andan aromatic motif proximal to the transmembrane domain. The presence ofat least four out of these five domains defines a viral envelope proteinas a Class I fusion protein. This retroviral transmembrane protein modelwas subsequently confirmed by structural determinations and mutationalanalyses (Chan et al., 1997; Kowalski et al., 1991; Weissenhorn et al.,1997). Common structural motifs are present not only in orthomyxovirusand retrovirus fusion proteins, but also in those of paramyxoviruses,filoviruses (such as Ebola virus, EboV) (Gallaher, 1996) andarenaviruses (Gallaher, DiSimone, and Buchmeier, 2001). The Gallaherstructural model of the EboV fusion protein (GP2) has also beenconfirmed by x-ray crystallographic methods (Malashkevich et al., 1999;Weissenhorn et al., 1998).

FIG. 1 shows the five, previously-described, domains of the fusionproteins of the six families of Type I viruses. The fusion proteinsoriginate in a hydrophobic fusion peptide, terminate in an anchorpeptide, and incorporate an extended amino terminal alpha-helix(N-helix, usually a “heptad repeat” or “leucine zipper”), a carboxylterminal alpha-helix (C-helix) (Carr and Kim, 1993; Suarez et al., 2000;Wilson, Skehel, and Wiley, 1981), and sometimes an aromatic motifproximal to the virion envelope. Also shown is the sixth domain, thefusion initiation region (FIR), discovered by the present inventors.

Fusion Inhibition in Type I Viruses.

Previous attempts by the present inventors (Garry) and others to designpeptides and peptide mimics, antibodies, and other factors that inhibitfusion in Type I viruses have focused on the fusion peptide, theN-helix, and the C-helix of the fusion proteins. In the case of fusionpeptides, analogs of the orthomyxoviruses and paramyxoviruses(Richardson, Scheid, and Choppin, 1980) and HIV-1 fusion peptide domains(Gallaher et al., 1992; Owens et al., 1990; Silburn et al., 1998) havebeen found to block viral infection, presumably by forming inactiveheteroaggregates. Peptides corresponding to portions of the N-helix andC-helix have also been found to be effective in inhibiting viralinfection both in vitro and in vivo. For example, a 17-amino-acidpeptide corresponding to the carboxy-terminal portion of the N-helix ofthe HIV-1 fusion protein, defined as the CS3 region, blocked HIVinfection (Qureshi et al., 1990). In addition, other N-helix and C-helixinhibitory peptides were developed based on the fusion proteinstructural model (Wild, Greenwell, and Matthews, 1993; Wild et al.,1992), including the C-helix anti-HIV-1 peptidic drug DP178 (T-20 orFUZEON®). DP178 overlaps the C-helix and the aromatic anchor-proximaldomain and inhibits HIV-1 virion: cell fusion at very low concentrations(50% inhibition at 1.7 nM) achievable in vivo following injection. In aclinical trial, 100 mg/day of DP178 caused an approximately 100-foldreduction in plasma HIV-1 load of infected individuals (Kilby et al.,1998). This result has greatly motivated the search for other HIV-1inhibitory peptides based on transmembrane protein structure (Pozniak,2001; Sodroski, 1999). Peptidic inhibitors of paramyxoviruses have alsobeen shown to inhibit viral replication (Lambert et al., 1996; Young etal., 1999). Studies by Watanabe and coworkers suggest that a similarapproach of targeting the N-helix and the C-helix of EboV GP2 may alsolead to useful inhibitors (Watanabe et al., 2000). Neutralizingantibodies directed against portions of the fusion protein domains havealso been shown to inhibit virion:cell fusion.

Observations in HIV-1.

A great deal of study has been devoted to fusion inhibition in humanimmunodeficiency virus HIV-1, one of the Type I RNA viruses. Bolognesiet al. (U.S. Pat. No. 5,464,933) and the present inventors (Garry, U.S.Pat. No. 5,567,805) teach that HIV-mediated cell killing can beinhibited by introducing peptides that bind to portions of thetransmembrane fusion protein of the HIV-1 virion. The Bolognesi DP178binding region, labeled FUZEON® in FIG. 7, lies primarily on the C-helixand is outside what is described in the present application the fusioninitiation region (FIR). Bolognesi demonstrates inhibition but teachesno method of inhibition. The present inventors (Garry) previouslydemonstrated inhibition at the CS3 region of HIV-1 TM, labeled CS3 inFIG. 7, but identified no method of inhibition, suggesting only thatCS3: CS3-receptor interaction is inhibited. The unexpected discovery ofthe FIR by the present inventors (as currently described herein) and thefact that the CS3 sequences lie within the FIR indicates that the CS3:CS3-receptor binding described in U.S. Pat. No. 5,567,805 is in factbinding that occurs between the CS3 portion of the FIR and portions ofthe cell membrane for which the CS3 portion of the FIR has an affinity.In addition, although Melikyan, Watanabe, Bewley, and others havedescribed fusion inhibition with introduced peptides, they have notexplained the mechanisms through which the inhibition occurs.Correspondingly, the location of the FUZEON® peptide is distant from theFIR, which strongly suggests that other elements of the fusion processoperate in the FUZEON® region.

In view of the foregoing, it is clear that there exists a need in theart for a more effective means for identifying those regions of virusesthat are involved in the infection process and for compositionseffective for preventing or inhibiting viral infection. The inventiondescribed and disclosed herein provides an effective solution to theseneeds.

SUMMARY OF THE INVENTION

Various embodiments of the instant invention provide for methods ofidentifying “factors” (compounds) capable of inhibiting membrane fusionbetween viruses and their host cells and, thereby, preventing orinhibiting infection of the host cell by the virus. Aspects of thisembodiment of the invention provide for methods of identifying theseinhibitory “factors” where the method comprises the steps of (a)identifying a virus having an envelope fusion protein having two, ormore, extended alpha helices, a fusion peptide, and a fusion initiationregion (FIR); (b) preparing a “target” wherein the target comprises theamino acid sequence of the FIR, (c) exposing the “target” to one or moretest compounds, and (d) identifying those test compounds that physicallyinteract with the “target”. For example, physical interaction can bedetected using a “target” bound to a solid substrate and a fluorescentlyor radioactively labeled test compound in a standard binding assay.Target and test compounds having dissociation coefficients (K_(d)) inthe micromolar range or lower (i.e. ≦about 9×10⁻⁶) are considered to bepositively interacting.

Other aspects of the instant invention provide for compositionscomprising an isolated peptide having the amino acid sequence of a viralfusion initiation region (FIR) or a functional segment of the FIR orhaving an amino acid sequence which is analogous to the sequence of aFIR or a functional segment of a FIR. As used herein, an analogous aminoacid or peptide sequence is a sequence containing a majority ofidentical or chemically similar amino acids in the same order as aprimary sequence. Such chemical similarities are well known to thoseskilled in the art.

Other aspects of this embodiment of the invention provide for isolated,typically substantially purified, peptides or peptide analogs that arecapable of preventing or inhibiting viral infection of a host celland/or inhibiting membrane fusion of a virus with a host cell, where thevirus comprises a membrane fusion protein having two (extended) alphahelices, a fusion peptide and a FIR.

Additional embodiments of the instant invention provide for methods oftreating or preventing viral infection by administering to a patient oneor more of the compounds identified by the methods described herein ascapable of inhibiting viral infection. In various aspects of thisembodiment of the invention the compounds administered are peptides orpeptide analogs comprising all or a functional segment of a viral FIRsequence. In any aspect of this embodiment of the invention theadministered compound is antigenic and is administered in an amountsufficient to eliciting an immune response.

Other embodiments of the instant invention provide for a “molecularfactor”, such as a plasmid, recombinant virus, or other substance whichenables or stimulates a cell or organism to produce a peptide or peptideanalog that is capable of preventing or inhibiting a viral infection ofthat cell or organism. In any aspect of this embodiment the “molecularfactor” is capable of preventing or inhibiting a viral infection whenadministered to a patient.

Another embodiment of the instant invention provides for antibodiescapable of inhibiting the virus:cell membrane fusion of a virus having afusion protein comprising two, extended alpha-helices, a fusion peptideand a FIR. In any aspect of this embodiment of the invention theantibodies are capable of binding specifically to amino acid sequencescomprising the FIR sequence, or fragments thereof of sufficient size toallow antibody recognition. Various aspects of this embodiment of theinvention provide for methods of producing the antibodies. In certainaspects of this embodiment, the method for producing antibodiescomprises: (a) providing as the antigen a peptide comprising a viralinitiation region (FIR) or an antigenic fragment of the FIR; (b)introducing the antigen in to an animal so as to elicit an immuneresponse; (c) collecting antibodies from the animal; and optionally, (d)purifying the collected antibodies to identify that fraction of thecollected antibodies having a high specificity for the antigen.

Other embodiments of the current invention provide methods of treatingpatients, which methods comprise administering to the patient antibodiesthat specifically recognize and bind to peptides comprising a FIR regionfrom a virus or comprising a functional fragment of such a FIR regionwhere the functional fragment is of sufficient size to allow itsspecific recognition by an antibody (that is, it is an antigenicfragment).

Other embodiments of the instant invention provide for methods ofproducing antibodies specific for FIR or functional fragments thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the domains of the fusion proteins of one member of each ofthese six viral families (namely, arenaviruses, coronaviruses,filoviruses, orthomyxoviruses, paramyxoviruses, and retroviruses). Thecircles in FIG. 1 show the approximate location of the FIR in each virusillustrated.

FIGS. 2 through 7 show the amino acid sequences of these fusion proteins(corresponding to SEQ ID NOs 16-21, respectively) and a schematicrepresentation of their ectopic structure. Specifically shown are thefive previously-described domains are the fusion peptide, i.e., theN-helix, the C-helix, the aromatic motif (if present), and the anchorpeptide. The newly-discovered sixth domain, the fusion initiationregion, or FIR is also identified. Each FIR is indicated by a polygon inFIGS. 2 through 7. The circled area behind the fusion proteins in eachof FIGS. 2-7 represents the primary virus:cell binding protein (VCBP) ofthe virus. The VCBP usually interacts with the portion of the fusionprotein which is most distal from the viral membrane and is thus shownto be so positioned in the Figures. Unlike the highly-conserved fusionprotein, the VCBP of each virus family is more divergent. It is usuallythe VCBP that dictates the host range of the virus and determines whichof the host's cell types are targeted for infection. The VCBP acts inthis capacity by recognizing and binding with specific cell surfaceproteins. The binding of the VCBP to the targeted cell proteins occursprior to and is typically a prerequisite for virus:cell fusion.

FIG. 8: Inhibition of coronavirus infectivity by fusion initiationregion peptides. Between 50 and 100 plaque forming units (PFU) of mousehepatitis virus strain A59 or SARS coronavirus strain Urbani werepre-incubated with or without the indicated peptides (approximately 100μM) in serum-free DMEM for 1 hour. Cells were then exposed topeptide-treated inoculum or a vehicle control (no peptide). After 1 houradsorption, the inoculum was removed, cells were washed twice with 1×phosphate buffered saline, and the cells were overlaid with DMEMcontaining 10% FBS and 0.5% agarose. Forty eight hours after infection,infected monolayers were fixed and stained with crystal violet todetermine plaque numbers.

FIG. 9: Inhibition of Lassa virus infectivity by fusion initiationregion peptides. Between 50 and 100 PFU Lassa virus was pre-incubatedwith or without the indicated peptides (approximately 100 μM) inserum-free BME for 1 hour. Cells were then exposed to thepeptide-treated inoculum or vehicle control (no peptide). After 1 houradsorption, the inoculum was removed, cells were washed twice with 1×phosphate buffered saline, and the cells were overlaid with BMEcontaining 5% FBS, 10 mM HEPES and 0.5% agarose. Four days afterinfection a second overlay containing 5% neutral red was applied, andplaques were counted 24 hours later.

ILLUSTRATIVE EMBODIMENTS OF THE INVENTION

The Sixth Domain of RNA Viruses Having Class I Membrane Fusion Proteins.

The arenaviruses, coronaviruses, filoviruses, orthomyxoviruses,paramyxoviruses, and retroviruses are the six families of RNA virusescurrently identified that have Class I membrane fusion envelopeproteins. The fusion proteins of these Type I viruses have previouslybeen shown by the present inventors (Garry) and others to incorporatefive conserved motifs, or domains (Carr and Kim, 1993; Gallaher et al.,1989; Suarez et al., 2000; Wilson, Skehel, and Wiley, 1981). Thesedomains comprise a fusion peptide, an N-helix, a C-helix, and anaromatic motif, all of which are ectodomains, and an anchor peptide,which is an endodomain.

Using computational analyses, secondary structure models, interfacialhydrophobicity calculations and other techniques, the present inventorshave made the surprising discovery of a highly conserved sixth domainthat is present in the fusion proteins of a wide variety of viruses(this sixth domain is described herein). The viruses possessing thisdomain include, but are not necessarily limited to the six classes ofRNA viruses listed above. To emphasize the critical function of thisnewly identified domain, which is an ectodomain, the domain is referredto herein as the fusion initiation region (FIR) of the viruses.

Various embodiments of the instant invention provide methods ofidentifying the FIR in arenavirus, coronavirus, filovirus,orthomyxovirus, paramyxovirus, and retrovirus families of viruses. Alsoprovided are methods of determining whether the FIR is present in otherknown virus families or in any newly discovered virus families.

As used herein the term “extended” alpha helix refers to an alpha helixhaving more than four “alpha helix turns” (specifically, more than 14amino acids).

Other embodiments provide for “factors” that the inventors haveunexpectedly found are effective for preventing or inhibiting viralinfection and/or virus:cell fusion.

As used herein the term “factors” includes, but is not limited toisolated peptides or functional peptide segments (or peptide analogsthereof) of the newly described fusion initiation region (FIR) domains,peptide mimics (“peptide mimic” refers to any compound or substance thatcould serve as a substitute for a peptide interacting with the FIR, thatis any compound that mimics the properties of a functional segment ofthe FIR), antibodies specific for functional FIR domains (e.g. idiotypicor anti-idiotypic antibodies) and other molecular compounds thatinterfere with virus:cell binding and/or fusion.

As used herein the term “functional segment” or “functional fragment” ofa fusion initiation region (FIR) refers to a fragment capable ofinhibiting virus:cell fusion, inhibiting viral infectivity, capable ofeliciting an antibody capable of recognizing and specifically binding tothe FIR and/or interfering with FIR-mediated cell infection.

As used herein, a “peptide analog” or “modified peptide” is preferablydefined as a FIR peptide modified to contain an amino group, an acetylgroup, a hydrophobic group (for example carbobenzoxyl, dansyl, ort-butyloxycarbonyl) or a macromolecular carrier group (for example lipidconjugate, polyethylene glycol, a carbohydrate or a protein) at theamino terminus. An additional class of FIR peptide analogs contains acarboxyl group, an amido group, a hydrophobic group or a macromolecularcarrier group at the carboxyl terminus. Other peptide analogs aredefined as FIR peptides wherein at least one bond linking adjacent aminoacids residues is a non-peptide bond (for example an imido, ester,hydrazine, semicarbazoide or azo bond), a peptide wherein at least oneamino acid residue is in a D-isomer configurations or a peptide in whichthe order of the amino acids is inverted. Additional peptide analogs areFIR peptides compromising at least one amino acid substitution wherein afirst amino acid residue is substituted for a second, different aminoacid residue (the amino acid substitution can be a conservedsubstitution or a non-conserved substitution). As used herein, suchpeptide analogs may comprise analogous amino acid sequences in which theanalogous sequences contain a majority of identical or chemicallysimilar amino acids in the same order as the primary sequences.

As used herein, the term “fusion initiation region” (FIR) generallyrefers to a region of a viral fusion protein involved in the initialstep or steps of viral infection and/or fusion with a host cell.

As used herein the term “peptide mimic” includes, but is not limited toorganic compounds or other chemicals that mimic the structure orfunction of the FIR peptide. Examples of peptide mimics include, but arenot limited to organic compounds comprising the functional side-groupsof an amino acid or peptide, but lacking the carbon/nitrogen backbone orpeptide bonds. Peptide mimic also refers to compounds that mimic theaction of these functional side-groups with other moieties.

Other molecules, such as idiotype or anti-idiotype antibodies orproteins selected via phage display methods, that bind to the peptides,peptide analogs or peptide mimics described in the present applicationmay also function as inhibitors of viral infection and/or virus:cellfusion. Also contemplated by the instant invention are plasmids, orrecombinant viruses, or other molecules or compounds that enable orstimulate the patient to produce an analog of the inhibitory compounds.For example, a recombinant protein, produced in an engineered bacterial,fungal, or mammalian cell, can be used to produce an immunogenic analogof the FIR of a viral fusion protein. Similarly, an anti-idiotypicresponse could be induced in the individual by using an engineeredprotein comprising a sequence corresponding to the binding site of aFIR-specific antibody.

As used herein the term “fusion peptide” preferably refers to ahydrophobic sequence at or near the amino terminus of a class I viralfusion protein (see, Gallaher et al., 1987; 1992).

As used herein the term “substantially purified” peptide or peptideanalog preferably refers to a peptide or peptide analog that is greaterthan about 80% pure. More preferably, “substantially purified” refers toa peptide or peptide analog that is greater than about 90% or greaterthan about 95% pure. Most preferably it refers to a peptide or peptideanalog that is greater than 96%, 97%, 98%, or 99% pure. Functionally,“substantially purified” means that it is free from contaminants to adegree that makes it suitable for the purposes provided herein. Methodsfor assessing purity are well known to those of skill in the art.Suitable methods include, but are not limited to gas chromatography (GC)linked mass spectrophotometry, high performance liquid chromatography(HPLC) analysis, and functional assays in cell culture systems that,inter alia, assess cytotoxicity.

As used herein the term “stable analog” refers to a peptide that has apharmacologically active half-life in biological systems. Biologicalhalf-lives of greater than 60 minutes are contemplated.

As used herein the term “peptide derivative” refers to a peptide thathas substituted amino acids different from those in the FIR sequence ofa viral fusion protein. Wherein the substitutions do not render thepeptide useless for the instant invention.

According to various aspects of the present embodiment of the inventionthe peptides, peptide analogs, peptide mimics, and other factors may beproduced by any means known in the art, including, but not limited to,chemical synthesis, recombinant DNA methods and combinations thereof.

The present invention provides methods for identifying the FIR of TypeI, and other, viruses and for treating or preventing infection by theseviruses. One possible mechanism by which the current invention may toprevent and/or inhibit infection is by interfering with the FIR mediatedvirus:cell fusion. The six families of RNA viruses now known to haveClass I membrane fusion proteins (Type I viruses) and representativemembers of each family are listed in Table 1 and Table 2.

TABLE 1 Representative RNA Viruses Having Class I Membrane FusionProteins (Type I Viruses) Family Representative Virus Shown in FIGURESArenaviruses Lassa Virus Yes Lymphocytic Choriomeningitis Virus (LCMV)No Junin Virus No Machupo Virus No Guanarito Virus No Sabia Virus NoCoronaviruses Severe Acute Respiratory Syndrome (SARS) Virus Yes MurineHepatitis Virus (MHV) No Bovine Coronavirus No Canine Coronavirus NoFeline Infectious Peritonitis Virus No Filoviruses Ebola Virus YesMarburg Virus No Orthomyxoviruses Influenza A Virus Yes Influenza BVirus No Influenza C Virus No Paramyxoviruses Measles Virus Yes MumpsVirus No Canine Distemper Virus No Newcastle Disease Virus NoRetroviruses Human Immunodeficiency Virus 1 (HIV-1) Yes HumanImmunodeficiency Virus 2 (HIV-2) No Human T-cell Lymphotrophic Virus 1(HTLV-1) No Human T-cell Lymphotrophic Virus 2 (HTLV-2) No HumanIntracisternal A-type Particle 1 (HIAP-1) No Human Intracisternal A-typeParticle 2 (HIAP-2) No

TABLE 2 Illustrated RNA Viruses Having Class I Membrane Fusion Proteins(Type I Viruses) shown in the Figures. Figure Family Virus Shown ProteinShown FIG. 2 Arenaviruses Lassa Virus GP2 FIG. 3 Coronaviruses SARSVirus S FIG. 4 Filoviruses Ebola Virus GP2 FIG. 5 OrthomyxovirusesInfluenza A Virus HA2 FIG. 6 Paramyxoviruses Measles Virus F1 FIG. 7Retroviruses HIV-1 TM

The sequences of illustrated Class I membrane fusion proteins (Type IViruses) shown in the figures are as follows: LASSA GP2 (GenbankAccession Number: A43492, amino acids 257-490), SEQ ID NO: 16; SARS S(Genbank Accession Number: AAQ9406, amino acids 864-1256), SEQ ID NO:17; EBOLA GP2 (Genbank Accession Number: AAM76034, amino acids 502-676),SEQ ID NO: 18; INFLUENZA HA2 (Genbank Accession Number: P03437, aminoacids 346-566), SEQ ID NO: 19; MEASLES F1 (Genbank Accession Number:VGNZMV, amino acids 116-553), SEQ ID NO: 20; HIV TM (Genbank AccessionNumber: AAB50262, amino acids 512-710), SEQ ID NO: 21.

Method of Identing the FIR.

Certain embodiments of the invention comprise a method of identifyingwithin the fusion proteins of viruses a conserved motif. The conservedmotif of the FIR regions from different viruses will have similarstructure and function. Additionally, the FIR regions of related virusesmay, but will not necessarily, have highly similar primary amino acidsequences. The current invention provides means for identifying theseregions, either with or without relying on their identity/similarity toknown sequences.

Other embodiments of the present invention provide for methods usefulfor preventing or inhibiting viral infection and/or virus:cell fusionusing peptides, peptide mimics, antibodies or other factors that aretargeted to the specific virus' FIR and interfere with the function ofthat FIR.

The FIR is typically between 50 and 100 amino acids in length, althoughit may be longer in some viruses. Various aspects of the currentembodiments provide methods for identifying the FIR of a viral fusionprotein wherein the methods comprises the following steps: (1) Thesequence of the fusion protein is first fitted to the HIV transmembranefusion protein scaffold, which comprises the N-helix, the C-helix, andother previously-described domains, in order to identify the N-helix andthe C-helix in the subject fusion protein. This fitting process isfacilitated by searching the primary amino acid sequence of the proteinfor two or more cysteines that have a propensity to form at least onecovalently bonded loop, which will be present in most but not all ofthese sequences. The N-helix can then be identified in the regionpreceding this cysteine loop by examining the region for charged aminoacids and other amino acids that have the propensity to form an alphahelix (e. g., glutamine (Q), alanine (A), tryptophane (W), lysine (K)and leucine (L)). (2) The amino terminus of the FIR is then identifiedon the N-helix. This terminus will usually lie within the final 10 to 20amino acids of the N-helix and will have a core typically comprisingthree or four hydrophobic amino acids (such as leucine (L) or alanine(A)), a positively-charged amino acid (such as lysine (K) or arginine(R)), a negatively-charged amino acid (such as glutamate (E)), and anaromatic amino acid (such as tyrosine (Y)). (3) The carboxy terminus ofthe FIR is then identified. In the case of all of the families exceptthe coronaviruses and paramyxoviruses, this terminus is thecarboxy-terminus of the first peptide sequence with positiveinterfacial-hydrophobicity that is found beyond the N-helix. Thisterminus is usually located beyond the cysteine loop, if the loop ispresent, and sometimes overlaps the C-helix or is positioned on theC-helix. The positive interfacial-hydrophobicity sequences have a highpercentage of aromatic amino acids (such as tryptophane (W),phenylalanine (F), and tyrosine (Y)) and small hydrophobic amino acids(such as glycine (G)). The degree of interfacial hydrophobicity of thesesequences can be determined by using the Wimley-White interfacialhydrophobicity scale, preferably with a computer program such as theMPEX program that incorporates this scale. “Interfacial hydrophobicity”is a measure of a peptide's ability to transfer from an aqueous solutionto the membrane bilayer interface and is based on the experimentallydetermined Wimley-White whole-residue hydrophobicity scale (Jaysinghe,Hristova, and White, 2000). Computer programs using this scale canidentify a peptide sequence of a peptide chain having positiveinterfacial hydrophobicity scores and are therefore the most likely toassociate with the surface of membranes. See Example 1, as an example ofthe application of this method to the identification of the FIR in theEbola virus.

In the case of the coronaviruses, which have longer alpha helices and agenerally larger scale, and the paramyxoviruses, in which the FIR isdiscontinuous because of a non-FIR sequence insert, the carboxy terminusof the FIR is the carboxy-terminus of the second peptide sequence withpositive interfacial-hydrophobicity that is found beyond the N-helix.The sequence between the N-helix and C-helix in the F1 protein ofparamyxoviruses is longer than the interhelical sequences of otherviruses with Class I viral fusion proteins. The F2 protein ofparamyxoviruses, which serves a receptor-binding function, iscorrespondingly shorter. Upon inspection of computer models, it isobvious to those skilled in the art that the F1 protein contains asequence insert between the N-helix and C-helix. Consequently, the FIRof paramyxoviruses contains two cysteine loops and twohigh-interfacial-hydrophobicity sequences and is discontinuous becauseadditional amino acids which are characteristic only of theparamyxoviruses and appear between the N-helix and the firsthigh-interfacial-hydrophobicity sequence are excluded from the FIR.

FIR Sequences.

The sequence of the fusion protein and FIR for each of the sixrepresentative viruses shown in FIG. 2 through FIG. 7 is given in therespective Figure and in the Sequence Listing provided below (SEQ ID NO:16 to SEQ ID NO: 21 provide the respective fusion proteins; and SEQ IDNO: 1 to SEQ NO: 7 provide the respective FIR). Although there is someminor sequence variation among the sister viruses within each of thesesix families, the FIR in any Type I virus can readily be identifiedusing the representative sequence given in the appropriate figure.

Methods of Inhibiting Fusion in these Viruses.

Other embodiments of the present invention provide methods of inhibitingvirus:cell fusion by interfering with the function of the FIR. Variousaspects of these embodiments include targeting the FIR with peptides,peptide mimics and other factors which may or may not be analogs of theFIR, in order to interfere with virus:cell fusion. In the variousaspects of this embodiment of the present invention the peptides,peptide mimics, and peptide analogs are between about 6 and 150 aminoacid residues long. More preferably, they are from about 8 to 50residues long, even more preferably they are from about 8 to 40 aminoacids in length or of such length as is necessary to provide effectiveinhibition of viral infection. As used herein the term “of such lengthas necessary to provide effective inhibition of the virus”, preferablyrefers to a length sufficient to provide a 5-fold or greater reductionin viral infectivity, when used according to the instant invention.Methods for quantifying reduction in viral infectivity are well known tothose of skill in the art. For example, reductions in viral activity maybe determined by plaque reduction, binding inhibition, titer reductionassays, or by animal challenge studies.

FIR peptides, peptides of analogous sequences, or fragments orderivatives thereof, contemplated as being part of the instant inventioninclude, but are not limited to, those comprising, as primary amino acidsequences, all or an efficacious part of one or more of the following:LASSA, X-LIMKNHLRDIMGIPYCNYSRYWYLNHTSTGKTLPRCWLI-Z (SEQ ID NO: 1); SARS,X-LIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQAAPHGVVFLHVTYVPSQERNFTTAPAICHEGKAYFPREGVFVFNGTSWFITQRNFFS-Z (SEQ ID NO: 2);EBOLA, X-LRTFSILNRKAIDFLLQRWGGTCHILGPDCCI-Z (SEQ ID NO: 3); INFLUENZA,X-IQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLF-Z (SEQ ID NO: 4), MEASLES,X-LGLKLLRYYTEILSLFG-Z (SEQ ID NO: 5) - - - - X-WYTTVPKYVATQGYLISNFDESSCTFMPEGTVCSQNALYPMSPLLQECLRGSTKSCARTLVSGSFGNRFILSQGNLIANCASILCKCYTTGTII-Z (SEQ ID NO: 6), wherein the “- - - -” indicatesthat the Measles FIR is discontinuous; and HIV,X-LQARILAVERYLKDQQLLGIWGCSGKLICTTAVP WNASWSNKSLE QIWNHTTWMEWD-Z (SEQ IDNO: 7). In each of the foregoing sequences the “X” and the “Z”respectively designate either the amino- or carboxy-terminus,respectively, of the peptide or an additional moiety, as describedbelow.

Other peptides provided by the instant invention include those havingthe sequence of a FIR region. In a preferred aspect of this embodimentthe FIR region is from a virus belonging to one of the viral familiesselected from the group consisting of arenaviruses, coronaviruses,filoviruses, orthomyxoviruses, paramyxoviruses, and retroviruses. In amore preferred aspect of this embodiment, the FIR is from a virusselected from the group consisting of Lassa Virus, LymphocyticChoriomeningitis Virus (LCMV), Junin Virus, Machupo Virus, GuanaritoVirus, Sabia Virus, Severe Acute Respiratory Syndrome (SARS) Virus,Murine Hepatitis Virus (MHV), Bovine Coronavirus, Canine Coronavirus,Feline Infectious Peritonitis Virus, Ebola Virus, Marburg Virus,Influenza A Virus, Influenza B Virus, Influenza C Virus, Measles Virus,Mumps Virus, Canine Distemper Virus, Newcastle Disease Virus, HumanImmunodeficiency Virus 1 (HIV-1), Human Immunodeficiency Virus 2(HIV-2), Human T-cell Lymphotrophic Virus 1 (HTLV-1), Human T-cellLymphotrophic Virus 2 (HTLV-2), Human Intracisternal A-type Particle 1(HIAP-1), and Human Intracisternal A-type Particle 2 (HIAP-2).

Other aspects of this embodiment of the invention provide for sequencescomprising a functional fragment of a FIR sequence or sequencesanalogous thereto, particularly from a virus belonging to one of theviral families selected from the group consisting of arenaviruses,coronaviruses, filoviruses, orthomyxoviruses, paramyxoviruses, andretroviruses (with the exception of the HIV-1 TM CS3 peptide previouslydescribed by the present inventors (Garry) and depicted in FIG. 7). Inanother preferred aspect of this embodiment, the peptide comprises afunctional fragment (except the HIV-1 TM CS3 fragment) or a sequenceanalogous to a functional fragment from a virus selected from the groupconsisting of Lassa Virus, Lymphocytic Choriomeningitis Virus (LCMV),Junin Virus, Machupo Virus, Guanarito Virus, Sabia Virus, Severe AcuteRespiratory Syndrome (SARS) Virus, Murine Hepatitis Virus (MHV), BovineCoronavirus, Canine Coronavirus, Feline Infectious Peritonitis Virus,Ebola Virus, Marburg Virus, Influenza A Virus, Influenza B Virus,Influenza C Virus, Measles Virus, Mumps Virus, Canine Distemper Virus,Newcastle Disease Virus, Human Immunodeficiency Virus 1 (HIV-1), HumanImmunodeficiency Virus 2 (HIV-2), Human T-cell Lymphotrophic Virus 1(HTLV-1), Human T-cell Lymphotrophic Virus 2 (HTLV-2), HumanIntracisternal A-type Particle 1 (HIAP-1), and Human IntracisternalA-type Particle 2 (HIAP-2).

As noted above the instant invention also contemplates derivatives ofthe FIR peptides described above and analogous sequences thereto. Thesederivative peptides may comprise altered sequences in which functionallyequivalent amino acid residues are substituted for residues within thesequence resulting in a silent change. For example, one or more aminoacid residues within the sequence can be substituted for by anotheramino acid of a similar polarity that acts as a functional equivalent,resulting in a silent alteration (e. g. substitution of leucine forisoleucine). Substitutes for an amino acid within the sequence may beselected from other members of the class to which the amino acidbelongs. For example, the nonpolar (hydrophobic) amino acids includealanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophanand methionine. The polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine. The positivelycharged (basic) amino acids include arginine, lysine and histidine. Thenegatively charged (acidic) amino acids include aspartic acid andglutamic acid. By way of further example, and not by way of limitation,such peptides may also comprise D-amino acids, and/or the may comprisean inefficient carrier protein, or no carrier protein at all.

FIR peptides may comprise peptides in which “X” comprises an aminogroup, an acetyl group, a hydrophobic group or a macromolecular carriergroup; and/or “Z” comprises a carboxyl group, an amido group ahydrophobic group or a macromolecular carrier group. Various aspects ofthe instant invention are drawn to peptides wherein the “X” moiety mayalso be selected from the group comprising: a hydrophobic moiety, acarbobenzoxyl moiety, dansyl moiety, or a t-butyloxycarbonyl moiety. Inany of the peptides of the instant invention the “Z” moiety may beselected from the group comprising: a hydrophobic moiety, at-butyloxycarbonyl moiety.

In other aspects of this embodiment of the invention the “X” moiety maycomprise a macromolecular carrier group. Such macromolecular carriergroup may be selected from the group comprising, but not limited to: alipid conjugate, a polyethylene glycol moiety, or a carbohydrate moiety.Similarly the “Z” may also comprise a macromolecular carrier group;wherein said macromolecular carrier is selected from the groupcomprising, but not limited to: a lipid conjugate, polyethylene glycolmoiety, or a carbohydrate moiety.

Various embodiments of this aspect of the invention also contemplatepeptides wherein one or more of the molecular bonds linking adjacentamino acid residues is a non-peptide bond. Such non-peptide bondsinclude, but are not limited to: imido, ester, hydrazine, semicarbazoideand azo bonds.

Yet other aspects of the instant invention provide for peptides whereinthe peptide comprises one or more amino acid residues that is/are in aD-isomer amino acid.

Other aspects of the instant invention provide for peptides comprisingone or more amino acid substitution wherein a first amino acid residueis substituted for a second, different amino acid residue, in thesequences provided above (or a functional segment thereof). In variousaspects of this embodiment, the amino acid substitution is aconservative substitution. In other aspects of this embodiment the aminoacid substitution is a non-conservative substitution. Yet other aspectsof this embodiment of the invention provide for peptides as describedabove except that one or more amino acid residues have been deleted.

In various preferred aspects of the instant embodiments the FIR peptidescomprise at least three contiguous residues of a FIR. More preferablythe FIR peptide comprises at least 8 contiguous residues of a FIR. Asused herein the term “FIR inhibitory peptide(s)” preferably refers to apeptide or peptides having the sequence of a FIR (or functional segmentthereof) and to such FIR peptides or functional segments in which one ormore amino acids is/are substituted for by functionally equivalent orchemically similar amino acids (see infra). It also refers toderivatives of these peptides, including but not limited to, benzylatedderivatives, glycosylated derivatives, and peptides that includeenantiomers of naturally occurring amino acids. In a preferred aspect ofthis embodiment the peptide is selected from those having the sequenceof any of SEQ ID NOs 1-7, 8-15, 22-25, and 30. In particularly preferredaspects of this embodiment the peptide has a sequence selected from thegroup consisting of SEQ ID NOs 22-25 and 30.

In yet other aspects of this embodiment of the invention, the FIRpeptides may be linked to a carrier molecule such as a protein,including but not limited to, human serum albumin (HSA).

Furthermore, the instant invention contemplates molecules comprising anycombination of the X and Z moieties and/or other peptide modificationsdescribed above.

Peptides according to the instant invention may be produced fromnaturally occurring or recombinant viral proteins. They may also beproduced using standard recombinant DNA techniques (e. g. the expressionof peptide by a microorganism that contains recombinant nucleic acidmolecule encoding the desired peptide, expressed under the control of asuitable transcriptional promoter, and the harvesting of desired peptidefrom said microorganism). In a preferred aspect of the invention, any ofthe peptides of the invention may be prepared using any chemicalsynthesis methodology known in the art including, but not limited to,Merrifield solid phase synthesis (Clark-Lewis et al., 1986, Science 231:134-139).

Embodiments of the instant invention also provide for other compoundsuseful for treating or preventing infection of a cell by a virus. Theseinclude antibodies (or active segments thereof, meaning portions ofantibodies capable of specifically recognizing a FIR region or afunctional segment thereof) and other molecules. Certain aspects of thisembodiment of the invention provide for antibodies that specificallyrecognize a FIR, or antigenic fragment thereof and/or are capable ofinterfering with virus:cell interaction sufficiently to prevent orreduce infection of the cell by the virus. Antibodies according to theseembodiments of the invention may be monoclonal or polyclonal.

Various embodiments of the invention provide for methods of producingantibodies capable of specifically recognizing a FIR and/or preventingor reducing infection of the cell by the virus. General methods forproducing antibodies are well known to those of skill in the art.Methods for producing antibodies according to the instant inventioncomprise the steps of (I) providing an antigen comprising a FIR or anantigenic fragment thereof (such antigen may be an unmodified peptide, apeptide mimic, a peptide analog, or a peptide derivative); (ii) exposingthe immune system of an animal to the antigen so as to induce an immuneresponse; (iii) collecting antibodies from the animal and identifyingthose antibodies that either specifically recognize a FIR (or functionalsegment thereof) and/or are capable of inhibiting or reducing virus:cellinfection in a dose responsive manner in assays that measure viralinfectivity.

Other embodiment of the instant invention provide for methods ofidentifying compounds capable of preventing or inhibiting infection by avirus comprising a FIR or that are useful as drug leads for thedevelopment of drugs for preventing or inhibiting viral infection. Suchmethods comprise the steps of: (I) identifying a virus having at leastone membrane fusion protein comprising a fusion initiation region thatis requisite for virus:cell fusion; (ii) preparing a target, where thetarget comprises the amino acid sequence of a FIR, or a functionalsegment of a FIR; (iii) screening a plurality of compounds to identifyat least one compound that binds to the target, thereby identifying atarget-binding compound; (iv) screening at least one target-bindingcompound to identify a target-binding compound that is capable ofspecifically preventing or reducing viral infection by the virus fromwhich the target was obtained or that us useful as a drug lead for thedevelopment of a drug for specifically preventing or reducing infectionby such a virus. As used herein the phrase “specifically preventing orreducing viral infection” means that the compound specifically preventsinfection by the target virus, without any substantial effect on anunrelated virus. For example, if a compound that specifically preventedinfection by the SARS virus would not prevent infection by the HIV-1virus.

As used herein the compounds (e. g. drugs or drug leads) identified bythe methods described above may be of any type, by way of non-exclusivelist they may be any peptide (or derivative, analog, or mimic thereof)this includes short peptides as are typically employed in phage displaylibraries, any antibody or active fragment thereof (i.e., any fragment,such as an Fab that is capable of specifically recognizing the target)or any other organic or inorganic molecule.

In any embodiment of the instant invention the FIR may be from any virushaving a membrane fusion protein comprising at least extended twoalpha-helices, a fusion peptide, and a fusion initiation region.Preferably, the virus is selected from a virus family, wherein the virusfamily is selected from the group consisting of: arenaviruses,coronaviruses, filoviruses, orthomyxoviruses, paramyxoviruses, andretroviruses. More preferably, the virus is selected from the groupconsisting of: Lassa virus, SARS (severe acute respiratory syndrome)virus, Ebola virus, influenza virus, measles virus, and HIV-1 (humanimmunodeficiency virus type 1).

According to various aspects of the instant invention, the peptidesand/or factors of the instant invention useful for treating orpreventing viral infection of a cell can target the amino acidssurrounding and within the FIR cysteine loop, the distal portion of theFIR N-helix, any of the interfacial hydrophobicity regions of the FIR,other areas of the FIR, or any combination of thereof. These factors,antibodies, peptides or peptide analogs (collectively compounds) may beused individually; alternatively they may be used in combinations of twoor more to prevent or inhibit infection of the cell by the virus. Themethods of preventing or inhibiting viral infection of the cell byinterfering with the function of the FIR provided by the instantinvention also include the use of neutralizing antibodies, producedexogenously or endogenously, against all or portions of the FIR. Thepurpose of such use is to interfere with the function of the FIR,thereby inhibiting viral infection of the cell and/or virus:cellmembrane fusion.

Other embodiments of the instant invention provide for compositions,including pharmaceutical compositions, comprising any and all of thecompounds, peptides (including analogs, derivatives, and mimicsthereof), antibodies, or any other molecule of the instant invention oridentified by the methods of instant invention. This includes, but isnot limited to, compositions containing any molecule that comprises,consists essentially of, or consists of a FIR, or a functional segmentof a FIR. It further includes, but is not limited to compositionscomprising any compound that specifically recognizes, binds to, orinterferes with the function of a viral FIR. As used herein, the phrase“interfering with the function of the FIR” means that a compoundinteracts with the FIR or with the cellular protein that serves as thereceptor that recognizes the FIR so as to prevent or reduce infection ofthe cell by the virus. Additionally, it is contemplated that thecompositions may comprise either one of the molecules described ormixtures of two or more of the molecules.

Further embodiments of the instant invention provide for methods oftreating or preventing infection of a cell by a virus (where the viruscomprises a FIR) using any of the compounds of the instant inventionand/or any compound identified by any of the methods of the instantinvention. Various aspects of this embodiment of the invention providefor administering an effective amount of any of the pharmaceuticalcompositions described herein to a patient suspected of being exposed toa virus (or having potential for being exposed to a virus) wherein thevirus comprises a FIR. In various aspects of the invention thepharmaceutical composition comprises an antibody that specificallyrecognizes and binds to a FIR (or functional segment of a FIR) or afragment of such antibody that specifically recognizes and binds to aFIR, or functional segment of a FIR.

The peptides of the invention may be administered to patients in anysterile biocompatible pharmaceutical carrier including, but not limitedto, saline, buffered saline, dextrose, and water. The peptides of theinvention may be administered using techniques well known to those inthe art. Preferably the agents are formulated. Techniques forformulation may be found in “Remington's Pharmaceutical Sciences”18^(th) ed., 1990 Mack Publishing Co., Easton, Pa. Methods ofintroduction include, but are not limited to, transdermal,intramuscular, intraperitoneal, intravenous, subcutaneous, oral andintramucosal administration, including intranasal administration. Fortransmucosal administration, penetrants appropriate to the barrier to bepermeated may be used. Such penetrants are generally known in the art.

Still other aspects of this embodiment of the invention provide formethods that comprise administering to a patient an effective amount ofa composition comprising at least one recombinant DNA or RNA molecule;where the RNA or DNA encodes a FIR (or functional segment thereof) or amolecule capable of specifically binding to a FIR or a cellular receptorthat recognizes a FIR so as to prevent or reduce infection by the virus.In a preferred aspect of this embodiment the recombinant RNA or DNAmolecule and or pharmaceutical composition further comprises theelements necessary to allow the protein encoded by the RNA or DNAmolecule to be expressed in a human cell. By way of non-exclusiveexample, in certain aspects of this embodiment of the invention therecombinant RNA or DNA molecule is part of a recombinant plasmid or arecombinant virus.

Example 1 Identification of the FIR in Ebola Virus

The method to identify the FIR of Class I viral fusion proteins can beillustrated by two examples. The first example is identification of theFIR in the minimal class I fusion protein glycoprotein 2 (GP2) of Ebolavirus, a filovirus. The boundaries of the N-helix and the C-helix ofEbola virus GP2 have been determined by x-ray crystallographic methods(Malashkevich et al., 1999). The terminal amino acids of the N-helixcontain the sequence ILNRKAIDF (SEQ ID NO: 8) that fits the consensus ofa core comprising three or four hydrophobic amino acids, apositively-charged amino acid, a negatively-charged amino acid, and anaromatic amino acid. Between these two helices are two cysteines in thesequence CHILGPDC (SEQ ID NO: 9). Defining the ends of the Ebola virusGP2 FIR is the sequence FLLQRWGGTCHILGPDCCI (SEQ ID NO: 10), which has aWimley-White interfacial hydrophobicity score of 2.59 as determined bythe MPEX program (Jaysinghe et al, 2002). Thus, the FIR of Ebola virusGP2 extends from amino acids 579 to 610.

Example 2 Identification of the FIR in Measles Virus

The second example is a complex class I fusion protein, the F1 proteinof measles virus, a paramyxovirus. The N- and C-helices of measles virusF1 can be identified by examining the primary sequence for amino acidswith the propensity to form helices. Alignment of the primary sequenceof measles virus F1 with the primary amino acid sequence of the F1protein of another paramyxovirus, Newcastle disease virus F1, can alsoaid in the identification of the helix boundaries. The structure of theNewcastle disease virus F1 protein has been determined by x-raycrystallographic methods (Chen et al., 2001). The boundaries of the N-and C-helices can thus be predicted to be amino acids 131-217 and455-491 respectively. In contrast to Ebola virus GP2 and most otherviral class I fusion proteins, the primary sequence between the N- andC-helices in the measles virus is longer than 100 amino acids. The FIRregion of measles virus F1 contains an insertion which, upon inspectionof computer models, is obvious to those skilled in the art, and thus theFIR structure is formed by a secondary arrangement that brings togethertwo parts of the primary sequence. The inserted sequence forms a loopexternal to the FIR. The terminal amino acids of the N-helix contain thesequence LKLLRYYTE (SEQ ID NO: 11) which fits the consensus of a corecomprising three or four hydrophobic amino acids, a positively-chargedamino acid, a negatively-charged amino acid, and an aromatic amino acid.There are eight cysteine residues in measles virus F1 between the N- andC-helices. On the basis of the alignment with Newcastle disease virus F1it can be determined that the first two cysteines and the second twocysteines form disulfide-linked loops. The first pair of cysteines inthe sequence, CTFMPEGTVC (SEQ ID NO: 12), is part of the FIR because itis bounded by a sequence WYTTVPKYVATQGYLISNF (SEQ ID NO: 13) with aWimley-White interfacial hydrophobicity score of 3.36, as determined bythe MPEX program. The second pair of cysteines in the sequence,CLRGSTKSC (SEQ ID NO: 14), is also part of the FIR because it isadjacent to a sequence TLVSGSFGNRFILSQGNLIANCASILCKCYTTGTII (SEQ ID NO:15) with a Wimley-White interfacial hydrophobicity score of 2.54, asdetermined by the MPEX program. Thus, the FIR of measles virus F1extends from amino acids 205 to 407, with amino acids 221 to 314representing an insertion that does not participate in FIR function.

Example 3 Identification of Coronavirus Fusion Inhibitory Peptides.Background

Severe acute respiratory syndrome (SARS) is a newly recognized illnessthat spread from southern China in late 2002/early 2003 to severalcountries in Asia, Europe and North America (Guan et al., 2004). SARSusually begins with a fever greater than 38° C. Initial symptoms canalso include headache, malaise and mild respiratory symptoms. Within twodays to a week, SARS patients may develop a dry cough and have troublebreathing. Patients in more advanced stages of SARS develop eitherpneumonia or respiratory distress syndrome. In the initial outbreakthere were 8098 cases worldwide, with an overall mortality of 9.6%. Apreviously unrecognized coronavirus (CoV) has been demonstrated to bethe cause of the new disease (Poutanen et al., 2003; Peiris et al.,2003; Drosten et al., 2003; Rota et al., 2003; Mara et al., 2003).Public health interventions, such as surveillance, travel restrictionsand quarantines, contained the original spread of SARS CoV in 2003 andagain appear to have stopped the spread of SARS after the appearance ofa few new cases in 2004. It is unknown, however, whether these draconiancontainment measures can be sustained with each appearance of the SARSCoV in humans. Furthermore, the potential of this new and sometimeslethal CoV as a bio-terrorism threat is obvious.

Coronaviruses are large positive-stranded RNA viruses typically with abroad host range. Like other enveloped viruses, CoV enter target cellsby fusion between the viral and cellular membranes, a process mediatedby the viral spike (S) protein. CoV S proteins, characterized to date,appear to consist of two non-covalently associated subunits, S 1 and S2.Using computational analysis, Garry and Gallaher (2003) first proposedthat the portion of the SARS-CoV S protein corresponding to the S2subunit fit the prototypical model of a class I viral fusion proteinbased on the presence of two predicted alpha helical regions at the N-and C-terminal regions of S2 (N-helix, C-helix) and an aromatic aminoacid-rich region just prior to the transmembrane anchor domain.

Materials And Methods.

L2 cells or Vero E6 cells were maintained as monolayers in completeDulbecco's modified Eagle's medium (DMEM) containing 0.15%HCO₃-supplemented with 10% fetal bovine serum (FBS), penicillin G (100U/ml), streptomycin (100 mg/ml), and 2 mM of L-glutamine at 37° C. in a5% CO₂ incubator. Mouse hepatitis virus (MHV) strain A59 or SARS CoVstrain Urbani or HK was propagated on L2 cells. For plaque assays, L2cells or Vero E6 cells were seeded at a density of 1×10⁶ cells in eachwell of a 6-well plate. Fifty to 100-plaque forming units (PFU) of MHVor SARS CoV were pre-incubated with or without approximately 100, mg/mlof peptide in serum-free DMEM for 1 hour. Cells were then infected withpeptide-treated inoculum or vehicle control inoculum. After 1 houradsorption, the inoculum was removed, cells were washed twice with 1×phosphate buffered saline, and the cells were overlaid with 10% FBS/DMEMcontaining 0.5% SEAPLAQUE® agarose (Cambrex Bio Science Rockland, Inc.,Rockland, Me.). Monolayers were fixed with 3.7% formalin and stainedwith 1× crystal violet 2 days post-infection, and plaque numbers weredetermined by light microscopy.

Results And Discussion.

Synthetic peptides corresponding to the FIR domains of the MHV or SARSCoV S protein were tested for their ability to inhibit infection bythese coronaviruses. The ability to inhibit formation of plaques in cellmonolayers is the most stringent in vitro test of a potential infectioninhibitor drug. Two peptides (GNHILSLVQNAPYGLYFIHFSW, SEQ ID NO: 22 andGYFVQDDGEWKFTGSSYYY, SEQ ID NO: 23) from the MHV FIR can inhibit plaqueformation by MHV, though the first MHV FIR peptide is more efficient(see FIG. 8A). Two peptides from the FIR of SARS, CoV(GYHLMSFPQAAPHGVVFLHVTY, SEQ ID NO: 24 and GVFVFNGTSWFITQRNFFS, SEQ IDNO: 25) inhibited plaque formation by this coronavirus (see FIG. 8B).There was also a significant reduction (−50%) in the average diameter ofthe residual plaques. These results suggest that this peptide inhibitsboth entry and spread of MHV. Similar results with these inhibitorypeptides were obtained in independent experiments, with 50% plaqueinhibition observed at concentrations of <5 μM. These results areunlikely to be explained by non-specific cytotoxic effects of thepeptides. Except for the plaques, cells in the monolayers were intactand viable. The low number of plaques grew were similar in size tocontrol plaques. Peptides from other regions also inhibited infection bythese viruses, but to a lesser extent than the most active FIR peptides(FIG. 8). For example, peptides from the fusion peptide region and thecarboxyl terminal helix (C-helix) of the MHV S and SARS CoV S providedsome inhibition (MHV S fusion peptide=MFPPWSAAAGVPFSLSVQY, SEQ ID NO:26; MHV S C-helix=QDAIKKLNESYINLKEVGTYEMYVKW, SEQ ID NO: 27; SARS CoV Sfusion peptide=MYKTPTLKYFGGFNFSQIL, SEQ ID NO: 28; SARS CoV SC-helix=AACEVAKNLNESLIDLQELGKYEQYIKW, SEQ ID NO: 29 Inhibitoryactivities in the, mM range were recently reported with coronavirusC-helix peptides by Bosch et al., (2003) and others (Bosch et al., 2004;Lui et al., 2004; Yuan et al., 2004; Zhu et al., 2004). However, no FIRcoronavirus inhibitory peptides have been reported. Nevertheless, inview of the current invention, the cited references collectively,provide support for the tremendous advantages of the currently disclosedand claimed inventions. That is, these references are consistent withthe inventors' assertion that the methods of the present invention canbe advantageously used to identify synthetic peptides that inhibitfusion/infectivity by members of the Coronaviridae family.

Example 4 Identification of Arenavirus Fusion Inhibitory Peptides.Background

Lassa fever is an often-fatal hemorrhagic illness named for the town inthe Yedseram River valley of Nigeria in which the first described casesoccurred in 1969 (Buckley and Casals, 1970). Parts of Guinea, SierraLeone, Nigeria, and Liberia are endemic for the etiologic agent, Lassavirus (LasV). The public health impact of LasV in endemic areas isimmense. The Centers for Disease Control, and Prevention (CDC) haveestimated that there are 100,000-300,000 cases of Lassa per year in WestAfrica and 5,000 deaths. In some parts of Sierra Leone, 10-15% of allpatients admitted to hospitals have Lassa fever. Case fatality rates forLassa fever are typically 15% to 20%, although in epidemics overallmortality can be as high as 45%. The mortality rate for women in thelast month of pregnancy is always high, about 90%, and LasV infectioncauses high rates of fetal death at all stages of gestation. Mortalityrates for Lassa appear to be higher in non-Africans, which is of concernbecause Lassa is the most commonly exported hemorrhagic fever. Becauseof the high case fatality rate and the ability to spread easily byhuman-human contact, LasV is classified as a Biosafety Level 4 and NIAIDBiodefense category A agent.

LasV is a member of the Arenaviridae family. The genome of arenavirusesconsists of two segments of single-stranded, ambisense RNA. When viewedby transmission electron microscopy, the enveloped spherical virions(diameter: 110-130 nm) show grainy particles that are ribosomes acquiredfrom the host cells (Murphy and Whitfield, 1975). Hence, the use for thefamily name of the Latin “arena”, which means “sandy”. In addition toLasV, other arenaviruses that cause illness in humans include Juninvirus (Argentine hemorrhagic fever), Machupo virus (Bolivian hemorrhagicfever), Guanarito virus (Venezuelan hemorrhagic fever) and Sabia virus(Brazilian hemorrhagic fever). Arenaviruses are zoonotic; each virus isassociated with a specific species of rodent (Bowen, Peters, and Nichol,1997). The reservoir of LasV is the “multimammate rat” of the genusMastomys (Monath et al., 1974). The wide distribution of Mastomys inAfrica makes eradication of this rodent reservoir impractical andecologically undesirable.

Signs and symptoms of Lassa fever, which occur 1-3 weeks after virusexposure, are highly variable, but can include fever, retrosternal, backor abdominal pain, sore throat, cough, vomiting, diarrhea, conjunctivalinjection, and facial swelling. LasV infects endothelial cells,resulting in increased capillary permeability, diminished effectivecirculating volume, shock, and multi-organ system failure. Frankbleeding, usual mucosal (gums, etc.), occurs in less than a third ofcases, but confers a poor prognosis. Neurological problems have alsobeen described, including hearing loss, tremors, and encephalitis.Patients who survive begin to defervesce 2-3 weeks after onset of thedisease. The most common complication of Lassa fever is deafness.Temporary or permanent unilateral or bilateral deafness occurs inapproximately 30% of Lassa fever patients during convalescence, and isnot associated with the severity of the acute disease. The antiviraldrug ribavirin is effective in the treatment of Lassa fever, but only ifadministered early (up to six days) in the course of illness (Johnson etal., 1987; McCormick et al., 1986). It is unknown whether ribavirin iseffective against other arenaviruses, such as Junin, Machupo, Guanaritoor Sabia viruses. No LasV vaccine is currently available.

Materials And Methods.

Vero cells were maintained as monolayers in Basal Medium Eagle (BME)containing 10 mM HEPES and 5% FBS. Lassa virus (Josiah strain) waspropagated on Vero cells. For plaque assays, Vero cells were seeded at adensity of 1×10⁶ cells in each well of a 6-well plate. Fifty to 100 p.f. u. of LasV were pre-incubated with or without peptide in serum-freeBME for 1 hour. Cells were then infected with peptide-treated inoculumor vehicle control inoculum. After 1 hour adsorption, the inoculum wasremoved, cells were washed twice with 1× phosphate buffered saline, andthe cells were overlaid with 2 ml of 0.5% agarose in BME containing 10mM HEPES and 5% FBS, and incubated for 4 days. A second overlaycontaining 5% neutral red was applied, and plaques were counted 24 hourslater.

Results And Discussion.

Synthetic peptides corresponding to the FIR domains of LasV glycoprotein2 (GP2) were tested for their ability to inhibit infection by thisarenavirus. A peptide (NYSKYWYLNHTTTGR, SEQ ID NO: 30) analogous to thesequence NYSRYWYLNHTSTGK from SEQ ID NO: 1 (LASSA FIR) can inhibitplaque formation by LasV (FIG. 9). A peptide analogous to another GP2region, the fusion peptide, (GTFTWTLSDSEGKDTPGGY, SEQ ID NO: 31) alsoinhibited infection by LasV, but to a lesser extent (FIG. 9). Noarenavirus inhibitory peptides have been reported. Collectively, theseresults suggest that our approaches can identify synthetic peptides thatinhibit fusion/infectivity by members of the Arenaviridae. Theseresults, in combination with our results with coronavirus FIR inhibitorypeptides, establish proof of the principle that FIR regions peptides canfunction as viral inhibitors

Each of the following documents is herein incorporated by reference:

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What is claimed is:
 1. A pharmaceutical composition for treating anongoing measles virus infection by inhibiting fusion of the virus with ahost cell membrane, the composition comprising a polypeptide in asterile biocompatible pharmaceutical carrier; wherein the polypeptideconsists of SEQ ID NO: 5, SEQ ID NO: 6, a segment of SEQ ID NO: 5 thatconsists of at least 8 consecutive amino acid residues thereof, and asegment of SEQ ID NO: 6 that consists of at least 8 consecutive aminoacid residues thereof.
 2. The composition of claim 1, wherein thepolypeptide includes a macromolecular carrier at the amino-terminusthereof, wherein the macromolecular carrier is selected from the groupconsisting of a lipid conjugate, a polyethylene glycol moiety, and acarbohydrate moiety.
 3. The composition of claim 1, wherein the sterilebiocompatible pharmaceutical carrier further comprises a penetrant. 4.The composition of claim 1, wherein the sterile biocompatiblepharmaceutical carrier comprises a buffered saline solution.
 5. Thecomposition of claim 1, wherein the polypeptide consists of SEQ ID NO:5.
 6. The composition of claim 1, wherein the polypeptide consists ofSEQ ID NO:
 6. 7. The composition of claim 1, wherein the polypeptideconsists of a segment of SEQ ID NO: 5 that consists of at least 8consecutive amino acid residues thereof.
 8. The composition of claim 1,wherein the polypeptide consists of an 8 to 40 consecutive amino acidsegment of SEQ ID NO:
 6. 9. A method for treating an ongoing measlesvirus infection by inhibiting fusion of the virus with a host cellmembrane, the method comprising: administering to a subject sufferingfrom a measles infection a polypeptide consisting of SEQ ID NO: 5, SEQID NO: 6, a segment of SEQ ID NO: 5 that consists of at least 8consecutive amino acid residues thereof, and a segment of SEQ ID NO: 6that consists of at least 8 consecutive amino acid residues thereof. 10.The method of claim 9, wherein the polypeptide includes a macromolecularcarrier at the amino-terminus thereof, wherein the macromolecularcarrier is selected from the group consisting of a lipid conjugate, apolyethylene glycol moiety, and a carbohydrate moiety.
 11. The method ofclaim 9, wherein the polypeptide is administered in a sterilebiocompatible pharmaceutical carrier.
 12. The method of claim 11,wherein the sterile biocompatible pharmaceutical carrier furthercomprises a penetrant.
 13. The method of claim 11, wherein the sterilebiocompatible pharmaceutical carrier comprises a buffered salinesolution.
 14. The method of claim 9, wherein the administering stepcomprises intranasally administering the polypeptide.
 15. The method ofclaim 9, wherein the polypeptide consists of SEQ ID NO:
 5. 16. Themethod of claim 9, wherein the polypeptide consists of SEQ ID NO:
 6. 17.The method of claim 9, wherein the polypeptide consists of a segment ofSEQ ID NO: 5 that consists of at least 8 consecutive amino acid residuesthereof.
 18. The method of claim 9, wherein the polypeptide consists ofan 8 to 40 consecutive amino acid segment of SEQ ID NO: 6.